PROJECT SUMMARY Stroke is the third leading cause of death and also the leading cause of serious long-term disability in the United States. About 87% of all strokes are classified as ischemic, which occurs as a result of an obstruction within a blood vessel supplying oxygen-rich blood to an area of the brain. Despite the efficacy and safety of thrombolysis (by tissue plasminogen activator, t-PA) and thrombectomy (by surgery), most patients are ineligible for treatment due to the narrow time-window. Thus, ischemic stroke is a huge unmet medical need; it's imperative to identify new stroke therapeutic targets. Swelling of astrocytes and neurons is a pathological hallmark of many neurological diseases, including ischemic stroke and traumatic brain injury. How it contributes to the pathogenesis remains unclear. Cell swelling activates the Volume- Regulated Anion Channel (VRAC), which typically facilitates regulatory volume decrease by mediating efflux of chloride and organic osmolytes, followed by release of osmotically obligated water. However, persistent VRAC activation in the brain is thought to be detrimental. For example, VRAC has been proposed to be a major pathway for the excessive glutamate release from swollen astrocytes, which over- stimulates neuronal NMDA receptors and causes excitotoxicity. Despite intense research in 3 decades, the molecular identity of VRAC was a longstanding mystery. Due to this gap, the previous evidence supporting VRAC's pathological role was mainly based on nonspecific pharmacological inhibitors. We developed an innovative high-throughput assay and through a genome-wide RNAi screen, have successfully identified a novel membrane protein SWELL1 (LRRC8A) as the only essential VRAC subunit. Our preliminary data showed that Swell1-dependent VRAC in astrocytes directly releases glutamate, which enhances neuronal excitability. Importantly, Swell1 astrocyte-specific KO mice were significantly protected from brain damage in transient middle cerebral artery occlusion (tMCAO) stroke model. This proposed research program will combine innovative approaches including cell and acute brain slice electrophysiology, live cell imaging, high-throughput chemical screening, cell-type specific KO mouse models, and in vivo experimental stroke models to elucidate the important role of the cell swelling- activated chloride channel in the pathogenesis of ischemic stroke. Completion of the proposed study will establish Swell1 channel as a key pathological mediator in stroke and provide a new ?druggable? ion channel target for not only stroke, but also other neurological disease associated with abnormal cell swelling.